Effects of Mutating CtrA-Binding Sites within the pile Promoter of Caulobacter Crescentus

Chong, Sarah (2017) Effects of Mutating CtrA-Binding Sites within the pile Promoter of Caulobacter Crescentus. Undergraduate thesis, under the direction of Patrick Curtis from Biology, University of Mississippi.

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Abstract

This project investigated the regulation of pilA by the global transcriptional regulator CtrA in the dimorphic bacterium Caulobacter crescentus. The pilA gene encodes the pilin subunits which are polymerized into helical filaments to produce pili, and the gene is directly activated by CtrA such that filaments are produced after cell separation. The pilA promoter harbors three regions that have been shown to bind CtrA (Binding Regions 1, 2, and 3), and multiple CtrA recognition sites are found in these regions, which is unusual for CtrA-dependent promoters. However, none of the binding sites match the CtrA consensus binding sequence. The canonical CtrA recognition sequence most closely matches the DNA sequence of Binding Region 1, and it is at relative variance with the sequences of Binding Regions 2 and 3. To examine the role of different binding sites in pilA activation, different pilA-lacZ constructs bearing isolated or combined binding region sites within the pilA promoter were introduced to C. crescentus. Ligation reactions were implemented to join novel pilA promoter constructs with cleaved reporter vectors (plac290). Heat shock methods were used to transform Escherichia coli with putative pilA-lacZ constructs, and electroporation was used to clone pilA-lacZ vectors into C. crescentus cultures. Beta-galactosidase assays were implemented to measure the strength of the novel pilA promoters in transgenic C. crescentus cells. My results demonstrate that the CtrA binding region bearing the binding site that is closest to the consensus sequence (Binding Region 1) barely induces pilA expression, and the pairing of this region with Binding Region 2 (whose binding sites are far from consensus) augments pilA expression - by more than twenty-fold - to levels which are characteristic of wild type. In addition, my results reveal that (1) the absence of Binding Region 3 enacts no effect on the intensity of pilA expression and (2) Binding Region 2 alone is not sufficient to induce significant pilA expression. In conclusion, my research indicates that the orchestration of multiple binding regions is necessary for effective transcription of pilA.

Item Type: Thesis (Undergraduate)
Creators: Chong, Sarah
Student's Degree Program(s): B.S. in Biology
Thesis Advisor: Patrick Curtis
Thesis Advisor's Department: Biology
Institution: University of Mississippi
Subjects: Q Science > QR Microbiology
Depositing User: Ms. Sarah Chong
Date Deposited: 22 May 2017 13:16
Last Modified: 22 May 2017 13:16
URI: http://thesis.honors.olemiss.edu/id/eprint/960

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